CN1638871B

CN1638871B - Method and apparatus for precise transfer and manipulation of fluids by centrifugal, and/or capillary forces

Info

Publication number: CN1638871B
Application number: CN038046431A
Authority: CN
Inventors: M·J·普吉亚; G·布兰肯斯泰因; R·-P·彼得斯; H·巴托斯
Original assignee: Siemens Healthcare Diagnostics Inc
Current assignee: Siemens Healthcare Diagnostics Inc
Priority date: 2002-02-26
Filing date: 2003-02-17
Publication date: 2010-12-29
Anticipated expiration: 2023-02-17
Also published as: KR101005799B1; HK1080023A1; US20090004059A1; WO2003072252A9; WO2003072252A1; EP1480750A1; CN1638871A; JP2005518531A; AU2003248353A1; US7459127B2; CA2477413A1; HK1080023B; JP4351539B2; US8337775B2; US20030166265A1; KR20040105731A

Abstract

A micro-liter liquid sample, particularly a biological sample, is analyzed in a device employing centrifugal and capillary forces. The sample is moved through one or more sample wells arrayed within a small flat chip via interconnecting capillary passageways. The passageways may be either hydrophobic or hydrophilic and may include hydrophobic or hydrophilic capillary stops.

Description

Method and apparatus by centrifugal force and/or accurate conveying of capillary force and operating fluid

Background technology

The present invention relates generally to the microfluid field, be applied to the analysis of various biological and chemical compositions.More specifically, the invention provides the centrifugal force of the surface property generation that utilizes passage in device and the method and apparatus that capillary force is analyzed.

For determine in body fluid or other fluids such as glucose, albumin or bacteria analysis thing have (or not existing) or its content, help the technical staff to finish analysis with reagent device usually.This reagent device contains one or more reagent areas, and the technical staff can apply sample fluid in these districts, then result and standard is compared.For example, reagent strip is immersed in the sample fluid, the reagent strip variable color, with the intensity of color or type and standard relatively with reference to colour chart.

When sample contained complicated composition, as many body fluid, this device just was difficult to preparation.Have to the composition of need affirmation or measurement was changed into suitable form before detecting by reagent, so that characteristic color to be provided.In the sample fluid other become branches to hinder anticipation reactions, they must be separated from sample, or with they on and.Sometimes the reagent composition is incompatible each other.In other cases, sample must be carried out preliminary treatment, to concentrate interested composition.These problems and other problems make the reagent composition that provides concrete chemical examination to need in the single assembly are provided.The prior art that comprises many device examples is intended that and overcomes these problems, and the ability of one or more special components in the analysing fluid sample is provided.

A kind of distinct methods is the series of steps that is prepared with analytical sample, but does not require that the technical staff does like this.An approach that realizes this point is a device of finishing the expectation operation automatically by preparing, but keeps reagent to isolate, and can avoid the problem of being discussed.For small sample, this analysis can be adopted the microfluid technology.

The microfluid device is very little, but they can receive sample, select requirement sample, dilution or washing sample, sample is separated into each composition, and react with sample or its composition.If the technical staff need carry out these steps with a large amount of samples in the laboratory, generally must manually finish necessary step, perhaps,, just need the equipment that moves sample and composition thereof and introduce reagent, cleaning fluid, diluent etc. if finish automatically.Yet the sample of biological assay is generally very little, so operating procedure must be carried out on very little equipment.The sample required size that laboratory equipment is narrowed down to about 0.02-10.0 μ L is infeasible, and has adopted distinct methods.By this feature of generation in plastics or other suitable substrates, and, make channel attached small container with micron-scale with another layer covering gained base material.This container can be included in and apply the preceding reagent that adds in the container of cover layer.Also can handle passage as required, make it can be wetting or nonwetting by sample to be tested.When vias inner walls was moistening, sample, sample constituents or other fluids can move in this passage by capillarity, or avoid them to move when fluid does not drench vias inner walls.Like this, the passage of capillary size can make fluid move, and can prevent that also fluid from moving, just as there being valve.Another method that passage by this micron-scale moves fluid is by centrifugal force, centrifugal force overcome can not wetting inwall resistance.This simple general picture that the microfluid device is provided of describing.Concrete application is provided in many patents, below will have mentioned partial monopoly.

United States Patent (USP) 6,143 provides the setting off a discussion of some principle of container that assembling is used for all kinds analysis and passage in 248, and other application examples of these principles can be at United States Patent (USP) 6,063, finds in 589.The microfluid device of describing in these two patents often is arranged to disc type, and rotates on the equipment that needed centrifugal force in various degree can be provided, and makes fluid move to another container from a certain container.Generally speaking, sample is placed position near pivot, and improve rotary speed gradually, the part of sample or sample is moved in pivot container far away.This patent has been described the sample of how to isolate ormal weight and has been analyzed, and sample and other fluids is mixed be used for cleaning or other purposes, and how sample is separated into various compositions.

Other patents have been described with electrode and have been moved fluid by electro-osmosis, and for example United States Patent (USP) 4,908, and 112.Caliper Technology Corporation has the representative patents about microfluid device aspect, and wherein fluid moves by electronic propelling.Representational example is a United States Patent (USP) 5,942,443,5,965,001 and 5,976,336.

At United States Patent (USP) 5,141, in 868, sample to be sent in the chamber with capillary force, the measurement of sample can be undertaken by the electrode that is arranged in this sample chamber.

The inventor also relates to and need be provided for immunoassay and nucleic acid chemical examination, for example reagent device of the detection of bacterial pathogens, protein, medicine, metabolin and cell.Their purpose is, when needing incompatible composition for given analytic process, and when need be before analyzing sample being carried out preliminary treatment, overcomes related problem.This way to solve the problem is different with afore-mentioned, hereinafter will describe in detail.

Summary of the invention

General features of the present invention is to adopt microfluid analysis of technology device, thereby the atom that carries out with improved procedure sample analyzing method is provided.Device of the present invention also makes the analysis of adopting the traditional analysis bar not carry out so far become possibility.

Analytical equipment of the present invention can be called " chip " here, and it is the thin plastics of small pieces normally, have wherein dug out the well that receives the microlitre size of liquid sample, and these wells interconnect by the capillary channel of the about 10-500 μ of width m, the degree of depth 5 μ m at least.Passage can be used known method, preferably by the plasma polymerization reaction at inwall, becomes hydrophobic or hydrophilic.Hydrophobicity or hydrophilicity are regulated on demand according to the performance of sample fluid to be tested.In certain embodiments, regulating hydrophobic surface avoids deposit attached on the inwall.In other embodiments, regulating water-wetted surface removes liquid basically fully.

Two types capillary bolt is disclosed, a kind of narrow bolt and a kind of wide bolt with hydrophilic inwall with hydrophobic inwall.In the chip basis part, form the feature of expectation, reagent is placed suitable well, apply top section then, thereby make chip.

In certain embodiments, analysis chip of the present invention comprises the hydrophilic capillary of the qualification section that is connected with the well of placing sample fluid.Sample fluid is filled this section by capillarity, thereby the sample of fixed volume is provided, to be transported to the analysis that other wells are expected subsequently.In certain embodiments, the capillary tube segment of qualification presents the U-loop shape that each end opening leads to atmosphere.In other embodiments, the capillary tube segment of qualification is linear.

By adopting a plurality of wells that connect by capillary channel, can provide and can carry out repeatedly the separately sample fluid of processing with predefined procedure, thus many problems of avoiding the traditional test bar to be difficult to overcome.For example, sample fluid can its with clean or preliminary treatment before suitable agent contacts.Can adopt more than one reagent in the reaction in turn to single sample.In addition, can from sample, remove liquid, react the certainty of measurement of carrying out on the sample to improve in the back that reacts.Makes to describe these structures and other possibility structures of explanation exemplary device of the present invention hereinafter with reference to accompanying drawing and institute.

The accompanying drawing summary

Fig. 1 is a kind of analytical equipment of the present invention.

Fig. 2 is second kind of analytical equipment of the present invention.

Fig. 3 a and 3b represent hydrophobic and hydrophilic capillary bolt.

Fig. 4 a represents multipurpose analytical equipment of the present invention.

The exemplary configuration that Fig. 4 b represents to adopt the Versatile apparatus of Fig. 4 a to provide to 4j.

Fig. 5 represents can analyze at most the analytical equipment of ten samples.

Preferred embodiment is described

Flowing in the microchannel

Adopt the general littler passage that uses the researcher more previous to propose of device of the present invention than this area.Particularly, the passage that adopts among the present invention has about 10-500 μ m, the width of preferably about 20-100 μ m, and the passage of the big order of magnitude of other people general employings.The minimum dimension of this passage believes it is about 5 μ m, because less passage can filter out composition to be analyzed in the sample effectively.Channel depth generally will be less than width.The applicant finds that except that initial flow, the passage in preferable range of the present invention can pass through capillary force, and without centrifugal force moving liquid sample.For example, can stop mobile by the treated capillary wall of dredging sample fluid that becomes.Resistance capillaceous can overcome by applying centrifugal force, then cancellation centrifugal force after liquid begins to flow.As selection, if the treated close sample fluid that becomes of capillary wall, fluid will flow without centrifugal force or other power by capillary force.If comprise hydrophilic bolt in this passage, just set up and flow by the power that applies the influence that overcomes hydrophilic bolt.The result can be according to the needs of the analysis of being carried out, and liquid can be measured and move to another district from a district of device.

Derived a Mathematical Modeling, it relate to rerum natura, the fluid of centrifugal force, fluid surface tension, capillary wall the surface can, particles contained surface energy in capillary size and the fluid to be analyzed.It can predict that fluid passes through flow capillaceous, and the hydrophobicity or the hydrophilicity of expectation.Can draw following General Principle from the relation of these factors.

For any given passage, the interaction of liquid and channel surface can or not produce appreciable impact to the mobile generation of liquid.When the ratio of the surface area of passage and volume is big, i.e. cross-sectional area hour, the interaction between liquid and conduit wall becomes clearly.This situation, when relating to nominal diameter less than the passage of about 200 μ m, and when can relevant capillary force playing a leading role with the surface of liquor sample and inwall, especially true.When inwall was moistening by liquid, liquid just moved in passage and need not to apply external force.On the contrary, when inwall not by liquid when moistening, liquid is just attempted to discharge from passage.These general trends can be used for causing liquid to move along passage, or make it stop to move in the junction with another passage with different cross-sectional.If liquid stops to flow, just by applying power moving liquid such as centrifugal force.As selection, can adopt other means that comprise air pressure, vacuum, electric osmose etc., these means can have the long-pending or surface of varying cross-section can passage between the junction induce and produce needed pressure and change.A feature of the present invention is that the passage that moves of liquid is littler than the passage that has adopted so far.Cause like this obtaining bigger capillary force, and can only pass through the capillary force moving liquid, and do not need external force, remove the nonessential capillary that overcomes short-term and stop up.Yet less passage is perhaps inherently to the obstruction sensitivity of particle in Biosample or the reagent.Therefore, the surface of conduit wall can be according to regulating in order to be used in the requirement that has on sample to be tested such as blood, the urine fluid for example.These characteristics make the design of analytical equipment more flexible.It is littler that this installs the dish that uses in the comparable prior art, and can use littler sample manipulation.Other advantages will become more obvious from the description of this device and embodiment.

Analytical equipment of the present invention

Analytical equipment of the present invention can be described as " chip ".They generally are little and flat, about usually 1-2 square inch (25-50mm ²).Volume of sample will be very little.For example, they can only hold about 0.3-1.5 μ L, thereby make the well broad of sample fluid and shallow, make sample be easy to see and measure by suitable equipment.Interconnective capillary channel will have 10-500 μ m, the width range of preferred 20-100 μ m, and its shape will be determined by the method that forms of passage.Channel depth will be at least 5 μ m.When limiting the sample of scheduled volume with capillary tube segment, the passage between the comparable reagent well of this capillary is big.

Can form capillary and sample wells though have such as several methods such as injection moulding, laser ablation, diamond lap or embossments, preferred employing injection moulding is to reduce the chip cost.The sample wells and the capillary network of general cutting generation expectation on the foundation of chip are made chip thereby cover top section then on the basis.

Chip often uses once and just abandons.Therefore chip should be made by cheap material as far as possible, and compatible with analytical reagent and sample.In most of example, chip will be by making such as plastics such as Merlon, polystyrene, polyacrylate or polyurethane, and as selection, they can be made by silicate, glass, paraffin or metal.

Capillary channel will be adjusted to have hydrophobic or hydrophilicity, and this performance is determined at the contact angle that the surface of solids forms by liquor sample or reagent.In general, if contact angle is just thought this surface hydrophilic, if contact angle is exactly more greatly hydrophobic less than 90 degree.Surface treatment can be become hydrophobic or hydrophilic.Preferably carry out the plasma induced polymerization reaction at channel surface.Analytical equipment of the present invention also available such as apply hydrophilic or hydrophobic material, grafting or sided corona treatment etc. be used to control the capillary wall surface can additive method make.In the present invention, preferably regulate the surface energy of capillary wall, i.e. hydrophily or hydrophobicity degree according to used sample fluid.For example, in order to prevent on the inwall of hydrophobic channel, to deposit, or in order to guarantee in the passage not residual liquid.

Liquid is stoped by the capillary bolt by motion capillaceous, and as what its title hinted, the capillary bolt prevents that liquid is along Capillary Flow.If capillary channel is hydrophilic and promotes liquid flow, just can adopt hydrophobic capillary bolt, promptly have the less passage of hydrophobic inwall.Liquid can not pass through hydrophobic bolt, because the combination of small size and inwall that can not be wetting causes the surface tension that stops liquid to enter.As selection,, between sample wells and capillary, just do not need bolt if capillary is hydrophobic.Liquid in the sample wells is prevented from entering capillary, until applying for example enough power of centrifugal force, makes liquid overcome the surface tension of opposition, and passes through hydrophobic channel.Characteristics of the present invention are only to need centrifugal force when liquid begins to flow.In case the hydrophobic channel inwall contacts fully with liquid, the power of opposition just reduces, because the existence of liquid has reduced the energy barrier relevant with hydrophobic surface.Therefore, liquid no longer needs centrifugal force to flow.Although do not need, in some cases, when liquid is mobile along capillary channel, perhaps be to continue to apply centrifugal force, easily to help rapid analysis.

When capillary channel is hydrophilic, liquid sample (supposition is a water-based) will flow in capillary naturally, and not need extra power.Capillary bolt if desired, a kind of selection is to use narrower hydrophobic fragment, and it can play aforesaid bolt effect.Also can use hydrophilic bolt, even also passable by hydrophilic capillary.This bolt is wideer than capillary, so the surface tension of liquid has just produced the less power that promotes liquid flow.If the wide variety between the bolt of capillary and broad is enough, liquid will stop in the porch of capillary bolt flowing.Have now found that liquid at last will be along the hydrophilic inwall wriggling of bolt, but by suitable design shape, this motion will be delayed fully, makes bolt effective, even inwall is hydrophilic also like this.A kind of preferred hydrophilic bolt is shown in Fig. 3 b, and the hydrophobic bolt (3a) of preamble description.

Fig. 1 represents to embody the testing arrangement of each side of the present invention.The for example sample of urine is placed reagent well R1.In this device, all passages are all handled and are hydrophobicity through plasma polymerization, make liquor sample can not move to R2 through passage under the situation that does not apply external force.When this device places on the platform, and with suitable speed rotation, when overcoming hydrophobic force, liquid sample just can move among the R2, and sample can react in R2 or other the preparation, is used for analysis afterwards.During filling R2, R3 also receives liquid, the amount that the sample that adds R1 can be received more than R2.R3 can provide the reaction second time of part sample, or the overflow of unnecessary sample only is provided.As selection, R3 can transmit pretreated part sample as required to R2.Because the passage between R2 and the R4 also is hydrophobic, must apply extra centrifugal force moving liquid sample.By applying centrifugal force, R5 can be full of with the sample of reaction from R4, maybe can be used for receiving and analyzing thing remaining liquid after R4 reacts and stays among the R4.If the measurement of product can be subjected to the influence of material in the liquid in addition among the R4, this step can provide improved measurement capability.In the design of Fig. 1, do not provide the capillary bolt, because capillary channel is hydrophobic.Yet, if passage is hydrophilic, exit at R1, R2 and R4 will provide the capillary bolt, thereby prevent that liquid from moving along capillary channel, till applying enough centrifugal force and overcoming bolt, enough centrifugal force overcomes after the bolt capillary force will be ordered about liquid sample and flow, and not need other centrifugal force.In other words, capillary force itself just is enough to mobile liquid sample.Should be noted that each well R1, R3, R4 and R5 have one to make when liquid sample is full of these wells that towards atmospheric access portal (V1, V2, V3 and V4) gas in the well can be discharged.

Fig. 2 represents to combine second kind of testing arrangement of metering capillary tube segment and hydrophilic bolt.Metering section guarantees to distribute the accurately liquor sample of amount, to improve analytical precision.Liquor sample is added sample wells R1, and this sample flows out from R1 by capillary force (passage is hydrophilic), and is full of the gauge rings L that is generally U-shaped.Shape shown in the shape of gauge rings capillaceous or section needn't have.Also can adopt straight line or linear capillary tube segment to replace.The end of ring is by V1 and V2 and environmental communication.Liquid sample moves to as far as hydrophilic bolt S1 (if desired, also can be hydrophobic bolt).When this device places on the platform, and when being enough to overcome the speed rotation of hydrophilic bolt resistance, contained liquid and moves among the reagent well R2 by capillary force just by bolt S1 among the sample ring L.Air just entered when liquid flowed out sample ring, thereby cut off the liquid at air entry point V1 and V2 place, and V1 and V2 define the length of liquid column, thereby define the amount that is transported to the sample among the reagent well R2.Be another reagent well R3 below the sample ring, it can be used for and the liquid sample reaction, or prepares the sample that is used for subsequent analysis, hereinafter will further describe.Because inwall is hydrophilic, liquid will move to R3 from R2 by capillary force.If capillary wall is hydrophobic, liquid will can not flow among the R3, till applying centrifugal force and overcoming opposite power.

Fig. 3 a and 3b represent to can be used for hydrophobic bolt (a) and the hydrophilic bolt (b) in the analytical equipment of the present invention.In Fig. 3 a, well R1 is full of liquid, and this liquid expands along the hydrophilic capillary that connects, and runs into narrow hydrophobic capillary channel and no longer mobile up to liquid, and the surface tension that this capillary provides has prevented that liquid from entering bolt.If apply active force from well R1 along capillary bolt direction, just can overcome opposite power, the liquid among the R1 can be transferred among the well R2.Equally, in Fig. 3 b, shown capillary bolt is hydrophilic bolt, and this bolt prevents that the liquid among the R1 from flowing among the well R2.The capillary bolt of this moment is not narrow, and has hydrophilic inwall.The increase of channel width avoids surface tension to cause that liquid flows out the capillary that is connected with the shape of bolt.Yet, as mentioned above, have now found that liquid will progressively wriggle along inwall, and overcome interception with one enough period.For great majority were analyzed purposes, bolt worked by its purpose, and is short because the sample analysis required time overcomes the prevention required time that is caused by moving naturally of liquid than liquid.

Fig. 4 a represents the plane of multipurpose analysis chip of the present invention.Form exhaust passage V1-V7, well 1-4 and 6-9, capillary bolt 5 in the chip, and U type sample ring L, dotted line represents to install the possible capillary channel that can form before the top cover in chip substrates.Obviously, many possible structures are arranged.Generally speaking, liquid sample can be added among the well R2, sample ring can be full of by capillary force, and be assigned among the well 6-8 by capillary bolt 5, sample can contact with reagent therein, and measures the reaction of

reagent.Well

1 and 3 can be used for preserving extra liquid sample, or as selecting, preserves the another kind of liquid that is used for the preliminary

treatment sample.Well

4 and 9 plays the effect in the chamber of preserving waste liq usually, or with regard to well 4, as sample fluid from the overflow well of well 2 or as the container of dress cleaning fluid.Each well can have suitable exhaust passage according to the requirement of being analyzed.Some possible structure is shown in Fig. 4 b to 4i.

In each figure of 4j, only finished some potential capillary channel at Fig. 4 b, other capillaries and well do not use.Exhaust line shown in Fig. 4 a is not shown, so that figure is more clear, but should be understood that if the analysis needs that carried out just can provide these passages.

In Fig. 4 b, liquid sample is added in the well 2, when applying enough centrifugal force (as selection, can adopt other means that overcome flow resistance) when overcoming flow resistance, sample just flows in the well 4 by hydrophobic capillary.Equally, by increasing the initial resistance that centrifugal force overcomes the hydrophobic capillary generation of connection, sample can flow through well 6,8 and 9 successively.

Well

4,6,8 and 9 can contain sample according to the requirement of the analytical method of expecting.

Fig. 4 c provides by the ability of hydrophilic bolt 5 from the liquid sample of ring L distribution and computation quantity, and the resistance of hydrophilic bolt overcomes by the centrifugal force that applies appropriate amount.As selection, extra sample can import in the well 4, and wherein sample is used agent treatment before importing well 6.Overcome hydrophobic resistance capillaceous by increasing centrifugal force, sample can be transported to well 8 and 9 successively from well 6.According to concrete analysis, well 6,8 and 9 can be used for making between molecule in the sample and the binding partner in the reagent well association reaction takes place, for example antibody and antigen, nucleotides and nucleotides, or the reaction of subject and object.In addition, in conjunction with to being coupled to certification mark or sign.

These wells also can be used for catching antibody, nucleotides or antigen in (capture) reagent well with being fixed to particle and lip-deep binding partner; Be used for cleaning or reaction and remove impurity, free material or interference; Or be used to add reagent, to proofread and correct or the control detection method.

One of them well generally produces and/or detection signal by the detection method that comprises in this well.The example of detection method comprises that Electrochemical Detection, spectral detection, magnetic detect, and by the detection of the reaction of enzyme, indicator or dyestuff.

Fig. 4 d provides the sample fluid of metered amounts has been transported to method well 6 and 8 from well 2 successively by gauge rings L and hydrophilic bolt 5.Sample delivery further before the reaction, can concentrated sample, or separating according to the needs of immunoassays and nucleic acid determination in the well 8 in well 6.In the said procedure modification, liquid can be transported to one of them exhaust passage from well 8.

Fig. 4 e and Fig. 4 d are similar, just replace well 6 and 8 with well 6 and 7.This program modification illustrates that also in order to carry liquid from well 6, linear array is not necessary.

Fig. 4 f and Fig. 4 d and 4e are similar, and wherein sample is carried by well 6,7 and 8 successively.

Fig. 4 g is a kind of modification, and wherein Ji Liang sample delivery arrives well 7, rather than Fig. 4 c is to the well shown in the 4e 6.

Fig. 4 h has illustrated a kind of chip, wherein sample fluid is added well 6, and is transported to well 8 by applying enough resistances of making every effort to overcome the clothes hydrophobic channel.According to the needs of being analyzed, reagent or buffer are joined the well 8 from well 3 and 4.Waste liquid is transported to well 9, can help improving the precision that reads of result in the well 8 like this.

Fig. 4 i has illustrated a kind of chip, wherein fluid sample is introduced well 1, and is transported to well 2, and there, as mentioned above, sample carried out preliminary treatment earlier before entering gauge rings.Secondly, overcome hydrophilic bolt 5, the preliminary treatment sample of metered amounts is assigned in the well 6 by applying centrifugal force.As the embodiment of front, connect hydrophobic resistance capillaceous by overcoming, sample can be transported in other wells, is in the well 9 in this case, further handles.

Fig. 4 j has illustrated a kind of device, and wherein sample adds well 3 rather than well 2.Well 2 receives cleaning fluid, and is transported to well 4 by the hydrophobic force that overcomes in the interface channel.By overcoming the resistance of hydrophilic bolt 5, the sample that well 6 receives from the metered amounts of U-shaped section.Can react in well 6, reacted sample delivery is further reacted to well 8, cleans with cleaning fluid then, and described cleaning fluid is transported to well 8 from well 4, arrives well 9 again.Read the color that manifests in the well 8 then.

Fig. 5 represents a kind of modification of chip of the present invention, wherein the single liquid sample is introduced among the sample wells S, and sample flows into the sample ring L1-L10 of ten the above-mentioned types from sample wells S by hydrophilic capillary under capillary drive.Should be understood that,, can provide any amount to replace ten sample ring according to chip size.Not shown exhaust passage among Fig. 5, but should be understood that they exist.Liquid is stopped by hydrophilic bolt in each ring.Then, when the power that applies when overcoming the capillary bolt, liquid can flow in the well and analyze.As shown in Figure 4, can produce many possible capillary channels arranges.

In many application, as described in following examples, measure the color that reagent and specimen reaction show.Same feasible is with the electrode of the The Small Well that is arranged in chip, and sample is carried out electrical measurement.The example of this analysis comprises the electrochemical signals converter based on ampere, impedance, electromotive force detection method.Example comprises oxidation and the detection of reduction chemistry and the detection of combination.

Embodiment 1

The preparation method who is used to detect the reagent of hemoglobin comprises: the at first coating solution and the coating ethanolic solution of the following composition of preparation:

Composition Concentration (mM)

Coating solution:

Glycerine-2-phosphate 200

Iron chloride 5.1

N (2-ethoxy) ethylene amine triacetic acid 5.1

Triisopropanolamine 250

Lauryl sodium sulfate [SDS] 28

Regulate pH value to 6.4 with 1N HCl

The coating ethanolic solution

Tetramethyl benzidine [TMB] 34.7

Diisopropyl benzene diperoxy hydrogen [DBDH] 65.0

4-methylquinoline 61.3

4-(4-diethylamino phenylazo) benzene sulfonic acid 0.69

4-(2-hydroxyl-(7, the 9-sodium disulfonate)-1-naphthyl azo) benzene 0.55

Then coating solution is applied on the filter paper (from the 3MM level of Whatman Ltd), and 90 ℃ of dry wet filter paper 15 minutes.Soak into dry reagent with the ethanol coating solution then, and under 90 ℃ dry 15 minutes again.

The preparation method who is used to detect albuminised reagent comprises:

The coating solution and the coating toluene solution that at first prepare following composition:

Composition Concentration (mM) Allowed band

Coating solution:

Aqueous solvent 1000mL-

Responsive buffer 93.8g (625mM) 50-750mM of tartaric acid cation

Quinaldine red background dye 8.6mg (20mM) 10-30mM

The coating toluene solution:

Toluene solvant 1000mL-

DIDNTB buffer 0.61g (0.6mM) 0.2-0.8mM

Lutonal M40 polymer reinforcing agent 1.0g 0.5-4g/L

DIDNTB=5 ', 5 "-dinitro-3 ', 3 "-two iodos-3,4,5, the 6-tetrabromophenol sulfonphthalein

Soak into filter paper with coating solution, use 204 and 237 Ahlstrom filter paper here, after soaking into for the first time with the aqueous solution, 95 ℃ of following dry filter papers 5 minutes, after soaking into for the second time with toluene solution, 85 ℃ dry 5 minutes down.

With following formulation test solution.Take by weighing protein and add in the MAS source of solvent.MAS solution is the PB that is designed to imitate the average behavior and the extreme performance of urine.The physical property of natural urine is shown in the following table:

Table A

Add 20.0mg Bovine albumin (Sigma Chemical Co. in 5mL MAS 1 solution in the 10mL measuring bottle, A7906), the albumin soln (2g/L=2mg/mL) of preparation 200mg/dL, reverberate then and static placement, up to the albumin complete hydrolysis, with MAS 1 volume adjustment is arrived 10.0mL then.

The Bovine hemoglobin of adding 10mg freeze-drying in 1L MAS 1 solution in the 1L measuring bottle (Sigma Chemical Co., H2500), the hemoglobin solutions (100mg/mL) of preparation 1.0mg/dL.

Downcut area 1mm ²Albumin and hemoglobin detect reagent, and place microfluid design shown in Figure 1, with 2mg/L albumin or 0.1mg/dL Hb test back observing response with the reagent well form of separating.Measure the reflectivity at 660nm place with digital processing device (Panasonic's 5100 serial digital cameras).Read the reflectivity that obtains when in device, adding behind the fluid contain urine and not contain albumin or hemoglobin one minute, the reactivity of its expression bar.

Storage 20 μ l samples in well R1 (chip design of Fig. 1), and transfer to well R2, then, by using Applied Motion Pruducts, Watsonville, CA. 513540 stepper motor drives able to programme that provide are centrifugal with 500rpm, connect the hydrophobic force of R1 in to R2 and R2 to the capillary of R4 to overcome, and transfer to R4.With before 5 μ l samples contact or contact back 1 minute, scribble the color of the filter paper of reagent among the measuring well R4.Analyzing the back transfers to liquid sample among the well R5 by the centrifugal force of 1000rpm.

2 images are got in each parallel test: an image is taken from preflood filter paper, and an image is taken from and injected the filter paper that cultivated 1 minute the back.Carry out four parallel tests.Also compare reagent paper is connected on the bar with the similar mode of traditional test bar.

Result among the table B:R4 on the hemoglobin reagent

Test	Sample	Hemoglobin in the sample	Observe
				1	Hb reagent on the bar	1mg/dl	Blue
1	Hb reagent among the R4	1mg/dl	Blue
				2	Hb reagent on the bar	0mg/dl	Orange
2	Hb reagent among the R4	0mg/dl	Orange

Hemoglobin reagent among the well R4 shows response clearly to hemoglobin from blank to 1mg hemoglobin/dL, identical with the situation of bar.Reagent filter paper manifests uniform color.Hemoglobin reagent among the R4 is soluble, and finds they to be cleaned out chamber R5.Repeated test, different is that hemoglobin reagent is placed well R2 rather than R4.

2 images are got in each parallel test: an image is taken from preflood filter paper, and an image is taken from and injected the filter paper that cultivated 1 minute the back.Carry out four parallel tests.

Result among the table C:R2 on the hemoglobin reagent

Test	Sample	Hemoglobin in the sample	Observe
				3	Hb reagent on the bar	1mg/dl	Blue
3	Hb reagent among the R2	1mg/dl	Blue
				4	Hb reagent on the bar	0mg/dl	Orange
5	Hb reagent among the R2	0mg/dl	Orange

Inject the preceding chip of liquid sample and have orange unreacted bedding and padding at well R2, colourless in R3 or R4.After injecting the hemoglobin sample, show the blueness of hemoglobin indicator dye among the R2.Test is transported to liquor sample among the well R4 to 1200rpm by improving rotary speed at last.

In another test, albumin reagent filter paper is placed well R4 and the retest that Fig. 1 designs.

2 images are got in each parallel determination test: an image is taken from preflood filter paper, and an image is taken from and injected the filter paper that cultivated 1 minute the back.Carry out four parallel tests.

Result among the table D:R4 on the hemoglobin reagent

Test	Sample	Hemoglobin in the sample	Observe
				3	Alb reagent on the bar	1mg/dl	Blue
3	Alb reagent among the R4	1mg/dl	Blue
				4	Alb reagent on the bar	0mg/dl	Orange
5	Alb reagent among the R4	0mg/dl	Orange

Inject the preceding chip of liquid sample and have the unreacted bedding and padding at well R4, colourless in R3 or R2 or R5.After injecting the albumin sample, show the blueness of albumin indicator dye among the R4.Test is transported to liquor sample among the well R5 to 1200rpm by improving rotary speed at last.

The all ingredients method that can replace the method among the above embodiment is arranged, and be used for chip of the present invention.The concentration of the analyte of measuring in the intensity that the variation of reagent makes the signal that produces and the clinical sample is directly proportional.These reagent comprise indicator dye, metal, enzyme, polymer, antibody and various other chemicals, and its drying is on carrier.Carrier commonly used is paper, film or the polymer with various sample picked-up and transportation performance.They can be introduced in the reagent well in the chip of the present invention, to overcome the problem of bringing with the reagent strip analysis.

All chemicals that can only comprise the color response needs that produce analyte with a reagent area of reagent strip.That the typical chemical reaction that takes place in the dried reagent strip can reduce is the dyestuff combination, that enzyme is urged, immunity, nucleotides, oxidation or reduction chemical reaction.In some cases, at a reagent layer 5 kinds of emulative and chemical reactions regularly take place at most, the method that detects hematuria is an example of the multiple chemical reaction that takes place in the single agents.The analyte detection reaction is based on the similar Peroxidase activity of hemoglobin, and it is by diisopropyl benzene diperoxy

hydrogen catalysis indicator

3,3 ', 5, the oxidation of 5 '-tetramethyl benzidine.In same bedding and padding, based on the catalytic activity of iron-HETDA complex, second reaction takes place and removes the ascorbic acid interference by the oxidation of diisopropyl benzene diperoxy hydrogen catalysis ascorbic acid in this complex.

Multiple reagent layer commonly used is measured a kind of analyte.The chemical reagent system is placed different reagent layers, and for to prepare such as Reaction Separation steps such as chromatography and filtrations.Full blood glucose bar multiple reagent district commonly used catches the intact erythrocytes that disturbs the floor that adds lustre to.Immunity-chromatogram law constitutes with the chemical reaction that takes place in the different reagent areas.The human body chorion promotes that glandular hormone (hCG) or albumin are exemplary application with bar of four reagent areas.First reagent at bar top is used for sampling and overlapping with next reagent area, creates conditions for parent sample (urine) is transported to first reagent area.The sample that to handle strides across the 3rd migration of agents then, and is wherein that reactant is fixing with the development color.This migration drives by the 4th reagent area of seizing excessive sample.The chromatography reaction takes place in the 3rd reagent area, and the 3rd reagent area is called test or trapping region, generally is the NC Nitroncellulose film.In first and second layers, the analyte response in distinctive antibody of analyte and the sample, and be transported to the NC Nitroncellulose film by the chromatogram mode.Antibody is bound on the colored latex particle and serves as a mark.If sample contains analyte, it will with the antibody response that is labeled.In capturing the district, when having analyte, SA just is fixed in the band of catching particle.Form painted p-wire.The second reagent band also is fixed in the trapping region, makes the reaction of control line and particle, forms color.When test system is suitably worked,, also tend on control line, form color even there is not hCG in patient's sample.Can become the chip of the present invention with reagent well to rapid analysis of this multistep, the reagent well has the analysis of suitable reagent to expect.

Above-mentioned albumin analysis also can be undertaken by additive method.Such as human serum albumin (HSA), gamma globulin (IgG) and Bence-Jones protein protein such as (BJP) mensuration that can in all sorts of ways.The simplest method is the dyestuff constraint method of the change color when depending on dyestuff constraint protein.Many dyestuffs have been adopted, example is 2-(4-hydroxyphenyl azo) benzoic acid [HAPA], bromocresol green, coeruleum bromocresolis, bromophenol indigo plant, tetrabromo phenol blue, pyrogallol red and two (3 '; 3 "-two iodos-4 '; 4 "-dihydroxy-5 '; 5 "-dinitrophenyl)-3,4,5,6-tetrabromophenol sulfonphthalein dyestuff (DIDNTB).Electrophoresis on various base materials has been used for from other Separation of Proteins albumins, then with albumin part dyeing, cleans subsequently and carries out photodensitometry.The example of dyestuff used herein is Ponceaux, crystal violet, amido black.For low concentration protein, i.e. protein in＜10mg/L albumin scope, the immunoassay such as immune turbidimetry commonly used.

Can adopt separation steps, wherein analyte in first well with reagent reacting, make reaction reagent flow to further reaction in second well then.Reagent can be resuspended in first well in addition, and moves to second well and react.Analyte or reagent can be captured in first or second well, and measure free reagent and the ratio that fetters reagent.

Free mensuration with constraint reagent is particularly useful for multizone immunoassay and nucleic acid determination method.There are various types of multizone immunoassays to be fit to the example of this device and conduct permission.Under the situation that adapts to the immune chromatograph determination method, reagent filter paper is placed well separately, and needn't contact by physics, because chromatogram power is not worked.Immunoassay or DNA determination method can develop into mensuration such as Gram-negative class (E.Coli for example, Entereobacter, Pseudomonas, Klebsiella) (for example StaphylococcusAureus Entereococc) waits bacterium with the Gram-positive class.Can develop immunoassay and be used for complete lath such as protein such as albumin, hemoglobin, myoglobulin, α-1-microglobulin, immunoglobulin (Ig), enzyme, glyoproteins, protease inhibitors and cytokines and peptide.Referring to for example: the multi-region analysis element with labelled reagent concentration district (Multizone Analytical Element Having Labeled ReagentConcentration Zone) of Greenquist in US 4806311, on February 21st, 1989; The employing of Liotta in US 4446232 contains the enzyme immunoassay (Enzyme Immunoassaywith Two-Zoned Device Having Bound Antigens) of the two district's equipment that fetter antigen, on May 1st, 1984.

Embodiment 2

The experiment of suspension again of dried reagent

Preparation:(0.1%w/w in the 0.1M PBS salt solution pH7.0) is distributed among the well R3 of chip design of Fig. 1, and in 40 ℃ vacuum drying oven dry 1 hour with 5 μ l phenol red solution.Before test, cover chip then with adhesive lid.MAS-1 cushioning liquid sample is placed well R1, and be transported among the well R3 by the centrifugal force under 500rpm as mentioned above.

Disperse and cover on the whole well R3 at phenol red after the drying.After filling R3 with the MAS-1 buffer, phenol red is almost suspended immediately again and can be removed from R3.

10 μ l phenol red stock solutions are distributed on the 3mm filter disc (OB filter), and dry in baking oven as mentioned above.After the drying filter disc is placed R2, fill R1 with the MAS-1 buffer then, and liquid is transported to well R2.

Chip is not painted before filling with liquor sample.Phenol red disperseed and cover whole well.Behind the filled R3 of MAS-1 buffer, phenol red is almost suspended immediately again, and can be transported to well R5 fully.

The potential application that dry reagent is dissolved as above embodiment again comprises:

● filter

● analysis by sedimentation

● cytolysis

● cell grade (mass discrepancy): centrifugation

● the enrichment (concentrating) of solid phase sample analysis thing (for example microballon) can be used to improve sensitivity.The microballon of enrichment can separate by the continuous centrifugal effect.

● available multichannel carries (metering in for example parallel and/or tactic all ingredients chamber) to obtain multichannel, and each passage produces a definite discrete results.Multichannel carry can by in the porch with fluid capillary system that be communicated with, that take into account a large amount of metering capillary loop, or measurement channel and/or system of being connected to the capillary bolt of each metering capillary loop realize.

● in chip design, can adopt and combining such as the auxiliary force of magnetic force etc.The carrier that captures as reagent or sample such as particle configuration example such as analyte or interfering materials such as magnetic beads.By such as density physical property separating particles such as (similar cracking fractionation).

Embodiment 3

Fig. 4 j has illustrated the chip that can be used to analyze

urine.Well

6 and 8 holds reagent used in the analysis, and well 3 is used to receive sample fluid, and well 2 is used to receive cleaning fluid.Well 3 is connected with hydrophilic sample ring L, and well 4 is connected with well 2 by hydrophobic capillary channel.

Well 6 holds and contains the protection and the fiber bedding and padding of buffer composition, the antibody that combines with the latex particle of blueness particularly for analyte (composition in the sample to be detected), and for the different antibodies of the analyte of using the fluorescein mark.In this embodiment, analyte is that the human body chorion promotes glandular hormone (hCG).Two kinds of antibody in it and the well 6 all react.

Well 8 holds the NC Nitroncellulose bedding and padding that irreversibly combine for the antibody of fluorescein.This antibody will react with the fluorescein that is transported to from well 6 well 8.

To urinate sample and add well 3, and be full of the hydrophilic capillary channel section between exhaust outlet V3 and the V4, and rest on hydrophilic bolt 5 places, thereby determine the sample to be analyzed of scheduled volume.Well 2 is full of the cleaning fluid of 0.6% saline solution such as buffering, with remove do not combine with the hCG analyte that comes artesian well 8 blue latex particle.Chip generally is about 500rpm with suitable speed, and rotation makes the sample of ormal weight flow in the well 6 by bolt 5.Cleaning fluid flows into the well 4 from well 2 simultaneously.

Through after the enough cultivation time, the composition in the bedding and padding in the well 6 suspends again, and two kinds of antibody all combines with analyte in the sample.Make chip with higher rpm (about 1000rpm) rotation then, liquid is transported to well 8 by the hydrophobic channel that connects well 6 and well 8 from well 6.

Pass through certain cultivation time again, the analyte antibody of fluorescein mark can be combined with the antibody at contained fluorescein in the well 8.Because analyte (hCG) all combines with two kinds of antibody, thereby the latex that makes blueness also combines with fiber bedding and padding in the well 8.Exist the blueness of indication analyte quantity this moment in the well 8, but in order to improve precision, clean this well.

With higher rpm (about 2000rpm) rotary chip for the third time, cleaning fluid is transported to well 8 from well 4, arrive well 9 then.Simultaneously all unconjugated liquid are transported to well 9 from well 8.After this step, the color in the well 8 can be measured by used camera apparatus among the embodiment 1 easilier.Color is directly proportional with the concentration of analyte in the sample, that is, be attached to well 6 on the analyte the quantity of blue latex particle be directly proportional.

Claims

1. device that distributes and analyze the uniform liquid sample comprises:

(a) sample wells of reception and the described liquor sample of transport portion;

(b) be communicated with the sample wells described in (a), receive hydrophily capillary channel from the described sample of described sample wells (a) by capillarity, described passage comprises the section of the volume that limits described uniform liquid sample, and described section is arranged between two openings that are communicated with atmosphere;

(c) hydrophily is carried capillary channel, its with described section of hydrophily capillary channel (b) with the first reagent well between liquid be communicated with, described hydrophily carries capillary channel to enter between two openings that are communicated with atmosphere described section, thereby makes air enter described section with alternative described uniform liquid sample from described two openings when described section even volume is transported in the described reagent well;

(d) be arranged in the described hydrophily transfer passage (c) to the hydrophily capillary bolt between the inlet of described section and described reagent well, described capillary bolt is used to prevent the conveying of described even sample, is used pressure, vacuum and electrodialysis and the means that apply power overcome up to the resistance of described bolt.

2. the device of claim 1 also comprises by hydrophily capillary channel and the described first reagent well at least one second reagent well with fluid connection.

3. the device of claim 2 also comprises the described second reagent well by hydrophily capillary channel and at least one claim 2 with at least one the 3rd reagent well of fluid connection.

4. the device of claim 1, the wherein said first reagent well contain be fit to described uniform liquid sample in the reagent of ingredient reaction.

5. the device of claim 4, the wherein said first reagent well contain be fit to described uniform liquid sample in the ingredient reaction, thereby produce the reagent of the response of the described component content in the described liquor sample of indication.

6. the device of claim 4, the wherein said first reagent well contain be fit to described uniform liquid sample in the ingredient reaction, thereby reduce the reagent of the interference of second kind of composition that described composition detects need.

7. the device of claim 4, the wherein said first reagent well contain the reagent that is fit to the described liquor sample of preliminary treatment.

8. the device of claim 5, the wherein said first reagent well contain be fit to described liquor sample in the composition reaction, thereby preparation feedback the reagent of composition.

9. the device of claim 8, the wherein said composition that has reacted in the second reagent well further reaction to indicate the content of the described composition in the described liquor sample.

10. the device of claim 1, wherein the inwall of the capillary channel described in (b) has and is adjusted to the water-wetted surface that can remove described liquor sample basically fully.

11. the device of claim 1 also comprises the electrode that is arranged in the described first reagent well, is used to measure the performance of described sample fluid.

12. the device of claim 2 also comprises the electrode that is arranged at least one described second reagent well, is used to measure the performance of described sample fluid.

13. the device of claim 3 also comprises the electrode that is arranged at least one described the 3rd reagent well, is used to measure described sample fluid performance.

14. a device that distributes and analyze the uniform liquid sample comprises:

(d) be arranged in the described hydrophily transfer passage (c) to the hydrophobicity capillary bolt between the inlet of described section and described reagent well, described capillary bolt is used to prevent the conveying of described even sample, is used pressure, vacuum and electrodialysis and the means that apply power overcome up to the resistance of described bolt.

15. the device of claim 14 also comprises by hydrophily capillary channel and the described first reagent well at least one second reagent well with fluid connection.

16. the device of claim 15 also comprises the described second reagent well by hydrophily capillary channel and at least one claim 15 with at least one the 3rd reagent well of fluid connection.

17. the device of claim 14, the wherein said first reagent well contain the reagent of ingredient reaction in suitable and the described uniform liquid sample.

18. the device of claim 17, the wherein said first reagent well contain ingredient reaction in suitable and the described uniform liquid sample, thereby produce the reagent of the response of the described component content in the described liquor sample of indication.

19. the device of claim 17, the wherein said first reagent well contain ingredient reaction in suitable and the described uniform liquid sample, thereby reduce the reagent of described composition to the interference of second kind of composition of need detection.

20. the device of claim 17, the wherein said first reagent well contain the reagent that is fit to the described liquor sample of preliminary treatment.

21. the device of claim 18, the wherein said first reagent well contain be fit to described liquor sample in the composition reaction, thereby preparation feedback the reagent of composition.

22. the device of claim 21, the wherein said composition that has reacted in the second reagent well further reaction indicating the content of the described composition in the described liquor sample.

23. the device of claim 14, wherein the inwall of the capillary channel described in (b) has and is adjusted to the water-wetted surface that can remove described liquor sample basically fully.

24. the device of claim 14 also comprises the electrode that is arranged in the described first reagent well, is used to measure the performance of described sample fluid.

25. the device of claim 15 also comprises the electrode that is arranged at least one described second reagent well, is used to measure the performance of described sample fluid.

26. the device of claim 16 also comprises the electrode that is arranged at least one described the 3rd reagent well, is used to measure described sample fluid performance.